Circadian Light Hygiene Is Associated with Anemia Markers in Young Adults

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

“Circadian Light Hygiene Associates with Anemia Markers in Young Adults”（ biology-3960631）

This manuscript aimed to explore the potential associations between circadian light pattern, physical activity, and sleep with anemia markers in young adults. The results revealed that larger normalized amplitude of BLE (NA BLE) positively correlated with higher HGB and MCH. An earlier
BLE acrophase (peak timing) was linked to higher MCH and smaller RDW-CV, indicating more uniform red blood cell size. Conversely, later acrophases of BLE and PA were associated with lower MCH, larger RDW-CV, and lower HGB, suggesting poorer hematological profiles in this population at higher risk of anemia. Later bedtimes also corresponded to lower HGB and MCH. These associations remained significant after adjusting for sex and age, although males generally had higher HGB and light exposure patterns of earlier phase and larger amplitude. Circadian profiles of BLE and PA weresignificantly correlated with hematopoiesis-related blood indices. Overall, this topic is very interesting and the current investigation expands the current research scope about the lighting and health into anemia field. This manuscript suits well for the special issue “Biological Rhythms and Molecular Clockworks in Physiology and Pathology”. However, some concerns appeared after reading the whole manuscript.

 

1. The “circadian light hygiene”should be defined first since the readers in other field might be not familiar with this concept.

 

2. Some important papers need to be reviewed and discussed, such as,

Rao, F., & Xue, T. (2024). Circadian-independent light regulation of mammalian metabolism. Nature Metabolism, 6(6), 1000-1007.

Li, A., Wei, X., Xie, Y., Ren, Y., Zhu, X., Liu, M., & Liu, S. (2024). Light exposure and its applications in human health. Journal of Biophotonics, e202400023.

Boyce, P. R. (2022). Light, lighting and human health. Lighting Research & Technology, 54(2), 101-144.

 

3. The latest prevalence of Anemia should be updated to inform the readers about the importance of this topic.

 

4. Since physical activity and sleep were also analyzed in the results, the readers might wonder why did you conduct the related analysis because both factors were not even mentioned in the introduction part.

 

5. How did you determine the sample size? Did you calculate the sample size needed before formal study?

 

6. For female participants, did you consider the menstrual characteristics？

 

7. For the lighting parameters, it had better to provide more details about the meaning of each parameter and how to calculate it rather than referring to previous research.

 

8. It had better to provide a figure to depict the lighting pattern to provide the readers more information about the current findings.

 

9. Please provide more details about ActTrust 2 device, such as the related validation references, and the manufacturer.

 

10. As far as I know, The ActTrust 2 device can record light intensity (lux) and its spectral components (infrared, red, green, blue, UVA, UVB, µw/cm2) every minute. Then why did you only focused on blue light?

 

11. All the scales or questionnaires need to be clearly identified their language versions and cite the related translation and validation articles.

 

12. Since there are many abbreviations in this manuscript, a list for abbreviations is needed.

Author Response

This manuscript aimed to explore the potential associations between circadian light pattern, physical activity, and sleep with anemia markers in young adults. The results revealed that larger normalized amplitude of BLE (NA BLE) positively correlated with higher HGB and MCH. An earlier BLE acrophase (peak timing) was linked to higher MCH and smaller RDW-CV, indicating more uniform red blood cell size. Conversely, later acrophases of BLE and PA were associated with lower MCH, larger RDW-CV, and lower HGB, suggesting poorer hematological profiles in this population at higher risk of anemia. Later bedtimes also corresponded to lower HGB and MCH. These associations remained significant after adjusting for sex and age, although males generally had higher HGB and light exposure patterns of earlier phase and larger amplitude. Circadian profiles of BLE and PA were significantly correlated with hematopoiesis-related blood indices. Overall, this topic is very interesting and the current investigation expands the current research scope about the lighting and health into anemia field. This manuscript suits well for the special issue “Biological Rhythms and Molecular Clockworks in Physiology and Pathology”. However, some concerns appeared after reading the whole manuscript.

 

Response 1: We are grateful to reviewer for thorough evaluation of our work and raising important questions.

 

The “circadian light hygiene” should be defined first since the readers in other field might be not familiar with this concept.

 

A2: We thank reviewer for this question. We now defined “circadian light hygiene” at first mentioning as “Circadian light hygiene can be defined as daily light exposure patterns, characterized by sufficient dynamic range and regularity to support circadian entrainment and prevent adverse health outcomes, often requiring strategic management in modern society”.

 

Some important papers need to be reviewed and discussed, such as,

 

Rao, F., & Xue, T. (2024). Circadian-independent light regulation of mammalian metabolism. Nature Metabolism, 6(6), 1000-1007.

 

Li, A., Wei, X., Xie, Y., Ren, Y., Zhu, X., Liu, M., & Liu, S. (2024). Light exposure and its applications in human health. Journal of Biophotonics, e202400023.

 

Boyce, P. R. (2022). Light, lighting and human health. Lighting Research & Technology, 54(2), 101-144.

 

A3: We appreciate this comment. We extended the Introduction, strengthening the discussion on light exposure's impacts on metabolism, health, and potential circadian disruptions by incorporating and discussing key recent studies (Rao & Xue, 2024; Li et al., 2024; Boyce, 2022). The revised text is as follows:

“Recent investigations underscore light’s multifaceted influence on health, encompassing metabolic regulation and circadian integrity. Specifically, light impacts mammalian metabolism independently of circadian rhythms through retinal and hypothalamic pathways, affecting glucose homeostasis and thermogenesis [5,6], with aberrant exposure potentially contributing to metabolic diseases. Light’s therapeutic applications include photobiomodulation for treating illnesses and modulating inflammation, potentially influencing micronutrient deficiencies implicated in anemia [3,7]. The effects of light, both positive and negative, on health are mediated through visual and non-visual systems, impacting circadian disruption and immune modulation, with outcomes contingent on individual age and physiological status [2-4, 8]. The pervasive nature of anemia, impacting approximately 25% of the global population [9], emphasizes its critical public health relevance. While recent data from the US reveals notable sex-related disparities, with prevalence at 13.0% in females compared to 5.5% in males [10], US prevalence is generally lower than global averages and also lower than in Russia, where sex-related differences are also observed [11].»

 

The latest prevalence of Anemia should be updated to inform the readers about the importance of this topic.

 

A4: Thank you for this comment. We added the following text to Introduction section:

“The pervasive nature of anemia, impacting approximately 25% of the global population (GBD, 2021), emphasizes its critical public health relevance. While recent data from the US reveals notable sex-related disparities, with prevalence at 13.0% in females compared to 5.5% in males (Williams et al., 2024), US prevalence is generally lower than global averages and also lower than in Russia, where sex-related differences are also observed (Bikbov et al., 2019).”

 

Since physical activity and sleep were also analyzed in the results, the readers might wonder why did you conduct the related analysis because both factors were not even mentioned in the introduction part.

 

A5: Thank you for this comment. We now added the following text to Introduction:

“Physical activity and sleep duration were also analyzed using actigraphy to explore their potential relationship with anemia. The inclusion of these factors was also motivated by their impact on general health and the recognition of their role in the pathogenesis of various conditions. Notably, studies have indicated a U-shaped association between sleep duration and anemia risk [18], and a dose-dependent decrease in depression risk with higher physical activity [19].”

 

How did you determine the sample size? Did you calculate the sample size needed before formal study?

 

A6: Our sample of 85 participants was selected via convenience sampling from young adult medical students at Tyumen State Medical University, Russia, to ensure a homogeneous cohort with minimal variability in age, lifestyle, and environmental factors—ideal for detecting subtle circadian associations. We deployed all 115 available actigraphs simultaneously during the same autumn month to avoid bias from changes in light exposure and ambient temperature. Of these, only 85 participants provided complete seven-day actigraphy data meeting inclusion criteria (e.g., valid wear time, no missing values). No a priori power calculation was performed, as this was an exploratory, observational study without predefined effect sizes for novel associations between circadian blue light exposure and hematological markers. However, post-hoc power analyses using G*Power software confirmed sufficient statistical power (>80%) to detect observed effect sizes (e.g., partial η² = 0.13 for NA BLE and MCH at α = 0.05). To address this in the manuscript, we propose the following addition in the Methods section (e.g., at the end of subsection 2.1. Study participants):

 

“Sample size was determined via convenience sampling from the accessible student population, utilizing available actigraphs simultaneously to minimize bias from environmental changes in LE / BLE and ambient temperature. No formal a priori power calculation was performed, as this was an exploratory study; however, post-hoc analyses confirmed adequate power (>80%) for the detected associations.”

 

For female participants, did you consider the menstrual characteristics？

 

A7: We appreciate this question. The study design prioritized maintaining a controlled ambient environmental factor for simultaneous weekly actigraphy in all participants to minimize external variability. This methodological approach, while crucial for the primary objective, made it impractical to collect and analyze detailed menstrual cycle data for female participants.

 

For the lighting parameters, it had better to provide more details about the meaning of each parameter and how to calculate it rather than referring to previous research.

 

A8: Thank you for this comment. We added clarifying information into Actigraphy section of the Methods as follows:

“ActStudio provides parametric estimates for all measured variables, including LE and BLE. For LE and BLE, these estimates include the MESOR, representing the average 24-h value for the fitted cosine curve; the Amplitude, quantifying the extent of daily variation; and the Acrophase, indicating the timing of the peak. The Normalized Amplitude of Blue Light Exposure (NA BLE) is a further informative index, calculated as the ratio of the 24-hour amplitude of the fitted cosine curve for BLE data to its MESOR. This normalization allows for standardized comparison of BLE patterns across individuals and was proofed to be an informative index of circadian light hygiene in the previous studies”.

 

It had better to provide a figure to depict the lighting pattern to provide the readers more information about the current findings.

 

A9: We appreciate this comment. We added text to the Results: “Average 24-Hour Patterns Light and Blue Light Exposure of the Study Participants.” and accordingly Supplemental Figure 1 which depicts Average 24-Hour Patterns of Light and Blue Light Exposure of the Study Participants.

 

Please provide more details about ActTrust 2 device, such as the related validation references, and the manufacturer.

 

A10: Thank you for your comment requesting more details on the ActTrust 2 device. To address this concisely, we now added to the existing description in the Methods section (e.g., subsection on actigraphy), focusing on the manufacturer and key validation points:

“Validation studies confirm PIM's high correlation (r > 0.85) with polysomnography for sleep-wake estimation [20], and its RGB sensor for blue light aligns [21] with CIE S 026:2018 standards for melanopic irradiance [22].”

 

As far as I know, The ActTrust 2 device can record light intensity (lux) and its spectral components (infrared, red, green, blue, UVA, UVB, µw/cm2) every minute. Then why did you only focused on blue light?

 

A11: We acknowledge the ActTrust 2 device’s ability to record a wide spectrum of light. Our specific focus on blue light exposure is due to its primary role in entraining circadian rhythms. Intrinsically photosensitive retinal ganglion cells (ipRGCs), which relay light information to the master circadian clock (SCN), are most sensitive to blue light. Therefore, blue light is the most potent signal for ipRGCs to influence circadian timing, making it a pivotal metric for actigraphy-based circadian research.

 

All the scales or questionnaires need to be clearly identified their language versions and cite the related translation and validation articles.

 

A12: The only scale used in this study was Morningness-Eveningness Questionnaire (MEQ) score. The Russian version of the MEQ has been validated [Putilov et al., 2005] and has been extensively used in numerous prior studies. The following text was added to accordingly Methods section:

“The Russian version of the MEQ has been validated [25] and widely used in previous studies [e.g. 26].”

 

Since there are many abbreviations in this manuscript, a list for abbreviations is needed.:

 

A13: Thank you for this suggestion. We now added the following list of abbreviations:

BLE: Blue Light Exposure, irradiance in the short wavelength range, measured by the Blue channel of the Condor AcTrust2 device.

BLE NA: Normalized Amplitude of Blue Light Exposure (ratio of the 24-hour amplitude of the fitted cosine curve for BLE data to its MESOR, used to standardize the dynamic range of blue light exposure relative to its average value.

BMI: Body Mass Index.

CBC: Complete Blood Count.

CIE: Commission Internationale de l'Éclairage (International Commission on Illumination, referenced in relation to melanopic irradiance standards).

FDR: False Discovery Rate (a statistical method used to correct for multiple comparisons, with a critical value of 0.1 in the study).

GBD: Global Burden of Disease (a comprehensive assessment of health loss due to diseases, injuries, and risk factors).

HCT: Hematocrit (percentage of blood volume made up by red blood cells).

HGB: Hemoglobin (amount of oxygen-carrying protein in blood).

HSC: Hematopoietic Stem Cells

IS: Inter-daily Stability (a non-parametric index measuring consistency of activity or exposure across days).

IV: Intra-daily Variability (a non-parametric index measuring within-day fragmentation of activity or exposure).

JBI: Joanna Briggs Institute (an organization that provides tools for evidence-based healthcare, including critical appraisal checklists).

L5: Least Active/Exposed 5-hour Period (the 5 hours of lowest values for physical activity or light exposure).

LE: Light Exposure (general light exposure, measured via actigraphy in lux).

M10: Most Active/Exposed 10-hour Period (the 10 hours of highest values for physical activity or light exposure).

MCH: Mean Corpuscular Hemoglobin (average amount of hemoglobin per red blood cell).

MCHC: Mean Corpuscular Hemoglobin Concentration (average concentration of hemoglobin in red blood cells).

MCV: Mean Corpuscular Volume (average size of red blood cells).

MEQ: Morningness–Eveningness Questionnaire (a 19-item self-report tool to assess chronotype, with scores ranging from 16 to 86; higher scores indicate morning type).

MESOR: Midline Estimating Statistic Of Rhythm (a rhythm-adjusted mean).

NR1D1: Nuclear Receptor Subfamily 1 Group D Member 1 (also known as REV-ERB).

PA: Physical Activity.

PIM: Proportional Integrative Mode (a method for estimating motor activity in actigraphy).

RBC: Red Blood Cells (erythrocytes; also referred to as red blood cell count in 10¹²/L).

RDW-CV: Red Cell Distribution Width - Coefficient of Variation (variation in red blood cell size, expressed as a percentage).

VIF: Variance Inflation Factors (used to evaluate potential multi-collinearity among variables in regression models).

WASO: Wake After Sleep Onset (time spent awake after initially falling asleep).

Reviewer 2 Report

Comments and Suggestions for Authors

1. Could you explain the terms "circadian light hygiene" and "normalized amplitude of blue light exposure" for readers who might not be familiar with these concepts?
2. What specific factors influenced the choice of medical students as the study population, and how might their distinct lifestyle characteristics affect the findings?
3. Can you provide additional information on the statistical techniques employed, especially concerning the multivariate regression models? How were potential confounding variables identified and managed?
4. How did you conclude that a sample size of 85 participants was adequate for the study? Were power analyses performed to justify this choice?
5. Considering the cross-sectional design of the study, how do you address potential limitations in establishing a causal relationship between circadian light exposure and hematological variables?
6. How might the results vary in populations outside of Tyumen, Russia, or during different seasons? What does this mean for the generalizability of your findings?
7. Can you discuss the proposed mechanisms through which circadian light exposure may affect hematopoiesis? Are there specific biological pathways or factors that you consider particularly significant?
8. How do your results align with existing research on circadian rhythms and hematological health? Are there any conflicting studies that should be acknowledged?
9. What future research do you recommend to further investigate the connection between circadian light exposure and hematological variables? Are there particular interventions or longitudinal studies you would suggest?
10. What practical advice can be drawn from your findings for individuals at risk of anemia, especially those with disrupted circadian rhythms, such as shift workers?

Author Response

Could you explain the terms "circadian light hygiene" and "normalized amplitude of blue light exposure" for readers who might not be familiar with these concepts?

 

A1: We thank reviewer for this question. As also suggested by another reviewer, we now defined “circadian light hygiene” at first mentioning as “Circadian light hygiene can be defined as daily light exposure patterns, characterized by sufficient dynamic range and regularity to support circadian entrainment and prevent adverse health outcomes, often requiring strategic management in modern society”.

We also provided an explanation of "normalized amplitude of blue light exposure" in Methods as follows:

“The Normalized Amplitude of Blue Light Exposure (NA BLE) is a further informative index, calculated as the ratio of the 24-hour amplitude of the fitted cosine curve for BLE data to its MESOR. This normalization allows for standardized comparison of BLE patterns across individuals and was proofed to be an informative index of circadian light hygiene in the previous studies”.

Furthermore, as suggested by another reviewer, we provided List of Abbreviations used in this paper.

 

What specific factors influenced the choice of medical students as the study population, and how might their distinct lifestyle characteristics affect the findings?

 

A2: Thank the reviewer for raising this point. Medical students were selected via convenience sampling for accessibility, allowing simultaneous actigraphy monitoring of 85 participants in a single autumn month in Tyumen, Russia, to minimize environmental variability in light exposure and temperature; their narrow age range (18–25 years) and shared location ensured homogeneity, reducing confounders like age or geography, with post-hoc power analysis confirming adequate (>80%) statistical power for associations despite no a priori calculation in this exploratory study. Their potentially irregular academic schedules may induce circadian disruptions (e.g., later bedtimes), which could enhance internal validity by reflecting real-world patterns in a young, anemia-risk population but limit generalizability to other groups; however, objective actigraphy captured these variations, and associations remained robust after adjustments for sex, age, and multiple comparisons, as well as sensitivity analyses. We suggest expanding the limitations section to address generalizability and propose future studies in diverse populations.

 

Can you provide additional information on the statistical techniques employed, especially concerning the multivariate regression models? How were potential confounding variables identified and managed?

 

A3. Thank you for requesting further details on our statistical approach. The following text was added to Methods / Data analysis:

“We analyzed associations between actigraphy (LE/BLE metrics) and CBC data using multivariate linear regression. Models were adjusted for sex and age. Potential confounders (PA, chronotype, BMI) were identified a priori and through exploratory analysis, then stepwise included if they affected coefficients >10% or improved model fit.”

 

How did you conclude that a sample size of 85 participants was adequate for the study? Were power analyses performed to justify this choice?

 

A4: As also requested by another reviewer, the answer is as follows: Our sample of 85 participants was selected via convenience sampling from young adult medical students at Tyumen State Medical University, Russia, to ensure a homogeneous cohort with minimal variability in age, lifestyle, and environmental factors—ideal for detecting subtle circadian associations. We deployed all 115 available actigraphs simultaneously during the same autumn month to avoid bias from changes in light exposure and ambient temperature. Of these, only 85 participants provided complete seven-day actigraphy data meeting inclusion criteria (e.g., valid wear time, no missing values). No a priori power calculation was performed, as this was an exploratory, observational study without predefined effect sizes for novel associations between circadian blue light exposure and hematological markers. However, post-hoc power analyses using G*Power software confirmed sufficient statistical power (>80%) to detect observed effect sizes (e.g., partial η² = 0.13 for NA BLE and MCH at α = 0.05). To address this in the manuscript, we propose the following addition in the Methods section (e.g., at the end of subsection 2.1. Study participants):

 

“Sample size was determined via convenience sampling from the accessible student population, utilizing available actigraphs simultaneously to minimize bias from environmental changes in LE / BLE and ambient temperature. No formal a priori power calculation was performed, as this was an exploratory study; however, post-hoc analyses confirmed adequate power (>80%) for the detected associations.”

 

Considering the cross-sectional design of the study, how do you address potential limitations in establishing a causal relationship between circadian light exposure and hematological variables?

 

A5: We acknowledge the reviewer’s concern regarding our cross-sectional design and its inability to establish causality. As detailed in the Study Limitations section, this study reports significant associations between circadian light exposure metrics (e.g., NA BLE, acrophase) and hematological variables (e.g., HGB, MCH, RDW-CV) within a homogeneous cohort of young adult medical students. We explicitly do not claim a causal relationship, recognizing that causation cannot be inferred from cross-sectional data. These findings highlight potential avenues for future longitudinal or experimental research to elucidate mechanisms, such as those involving erythropoiesis or circadian regulation of stem cells. This limitation has been transparently incorporated into the manuscript (Study Limitations, last sentence: “Additionally, the cross-sectional design precludes causal inferences, necessitating longitudinal research to elucidate time-related relationships.”

 

How might the results vary in populations outside of Tyumen, Russia, or during different seasons? What does this mean for the generalizability of your findings?

 

A6: We acknowledge the reviewer’s valid query regarding the generalizability of our findings outside Tyumen, Russia, or across different seasons. As detailed in the Study Limitations, our results are specific to young adult medical students in Tyumen (~57°N latitude) during autumn (a relatively low-light period). Circadian light exposure (e.g., BLE patterns) is photic-environment dependent, thus associations with hematological variables (HGB, MCH, RDW-CV) may differ in seasons with longer daylight or at other latitudes (e.g., equatorial regions). The narrow age range further restricts applicability to broader demographics. We recommend longitudinal studies across seasons and locations for validation, a limitation are now transparently noted in the manuscript.

 

Can you discuss the proposed mechanisms through which circadian light exposure may affect hematopoiesis? Are there specific biological pathways or factors that you consider particularly significant?

 

A7: We appreciate the reviewer’s query regarding the mechanistic links between circadian light exposure and hematopoiesis. We propose that robust circadian light cues (e.g., high NA BLE, earlier acrophase) indirectly modulate erythropoiesis by synchronizing bone marrow circadian clocks. Key factors are NR1D1 (linking circadian rhythms to heme metabolism and RBC maturation) and sympathetic pathways impacting HSC rhythms. Brain iron homeostasis’s interaction with clock genes also plays a role. These pathways suggest indirect photic modulation of erythrocytic indices via circadian synchronization. Future research incorporating melatonin and EPO measurements is warranted to confirm causality, particularly regarding light’s impact on EPO production.

 

How do your results align with existing research on circadian rhythms and hematological health? Are there any conflicting studies that should be acknowledged?

 

A8: We appreciate the reviewer’s query regarding the alignment of our findings with existing research on circadian rhythms and hematological health. As noted in our Discussion, specific studies are scarce as was noted in a recent meta-analytic review (Busza et al., 2024, a newly added reference). Our results support prior research demonstrating pronounced morning peaks in HGB and HCT (e.g., Sennels et al., 2002; Busza et al., 2024 meta-analysis). Conversely, MCH and RDW-CV exhibit minimal diurnal variation, consistent with our hypothesis of indirect photic modulation via circadian entrainment rather than direct rhythmic shifts. Furthermore, we added the following text to Discussion:

“Currently, there is a limited scope of studies characterizing the circadian rhythms of CBC components as evident from a recent review [27], particularly in relation to environmental light cues, which underscores the novelty of our investigation into how circadian light hygiene modulates these variables.”

 

What future research do you recommend to further investigate the connection between circadian light exposure and hematological variables? Are there particular interventions or longitudinal studies you would suggest?

 

A9: We thank reviewer for this suggestion. As recommended we now added the following text to Discussion:

“Future research should prioritize longitudinal studies to establish causality. Multi-month prospective cohorts tracking circadian LE/BLE patterns alongside serial CBC components in populations prone to light disruption (e.g., shift workers, high-latitude residents) can reveal long-term effects on erythropoiesis and anemia risk. Specifically timed bright blue-enriched light to optimize BLE may directly test whether improved circadian light hygiene enhances erythrocytic indices and NR1D1-mediated heme regulation. Mechanistic studies incorporating biomarkers like melatonin and ferritin are also warranted to elucidate photic cue-hematopoiesis pathways.”

 

What practical advice can be drawn from your findings for individuals at risk of anemia, especially those with disrupted circadian rhythms, such as shift workers?

 

 

A10: We thank reviewer for this suggestion. As recommended we now added the following text to Discussion:

“Our findings suggest that individuals at risk of anemia, particularly those with low daylight exposure and disrupted circadian rhythms (e.g., shift workers), can optimize their circadian light hygiene. Seeking bright blue-enriched light exposure earlier in the day can advance light acrophase and increase normalized amplitude.”

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have studies several markers associated with anemia which is quite interesting. I have following recommendations

• Abstract should be modified to follow a standard format of providing background, methods, results and conclusion. These headings are optional but it should follow this basic idea of why this topic is important, how did you study it, what were the results and then what were conclusions.
• Authors should include critical appraisal tool for their study which will improve the trasparency. Either a JBI tool https://jbi.global/critical-appraisal-tools or a ROBINS-I tool. This enhances the quality of the study. They can include it as a table or supplementary material
• Why have authors dived deeper on the reticulocyte count or number of immature cells if there is a theory that it affects Hb production/erythropoesis. Please expand on this or re-do analysis to include reticulocyte count
• Was electrophoresis done in the study? Will that help? Please include this info in the discussion
• Many of the terms used in the article may be new to the readers unfamiliar with your research. Like what is acrophase? New readers may not be aware of these terms. I recommend to include a glossary of such terms as a supplementary file
• Discussion does a good job at what other studies "were about" but lacks in clarity of what other studies "found". Revisions on these aspects should be made
• Conclusion is written in a way that keeps everything ambiguous. It should be written in the greatest clarity since authors rely on it. If conclusion is written in a confusing manner, readers will be repulsed and avoid reading the entire article. It should be worded conclusively, "Circadian hygiene improved Hb although <very brief 1-2 limiting points>". Feel free to use your own language but I believe the conclusion needs to be entirely modified. 

Comments on the Quality of English Language

needs major language edits and improvement in clarity

Author Response

Authors have studies several markers associated with anemia which is quite interesting. I have following recommendations

 

A1: We are grateful to reviewer for thorough evaluation of our work and an encouraging feedback.

 

Abstract should be modified to follow a standard format of providing background, methods, results and conclusion. These headings are optional but it should follow this basic idea of why this topic is important, how did you study it, what were the results and then what were conclusions.

 

A2: We appreciate this comment. We now updated Abstract as follows:

“Background: Light exposure (LE) and its influence on circadian rhythms are recognized to impact various physiological domains, yet their specific associations with hematological status, particularly in high-latitude environments, remain under-explored. Understanding these links could offer insights into maintaining hematological health. Methods: This study investigated the relationship between 24-hour light exposure (LE), blue light exposure (BLE), sleep, and physical activity (PA) with hematological markers in 85 young adults (18–25 years). Participants underwent simulatenous 7-day monitoring using actigraphy and RGB sensors, coupled with morning blood sampling for hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), and red blood cell distribution width (RDW-CV). Results: Univariate analyses revealed significant correlations: normalized BLE amplitude (NA BLE) positively correlated with HGB (r = 0.369, p = 0.001) and MCH (r = 0.378, p < 0.001). A later BLE acrophase correlated with lower HGB and MCH, but higher RDW-CV. Later PA acrophase associated with lower MCH and higher RDW-CV, while later bedtime correlated with lower HGB and MCH. Multivariate regressions confirmed that a larger NA BLE predicted higher HGB (β = 0.206, p = 0.037) and MCH (β = 0.377, p < 0.001), and an earlier BLE acrophase predicted higher MCH and smaller RDW-CV. Conclusion: Advantageous circadian patterns of BLE and PA are linked to a favorable hematological status in young adults during light deficient fall season at higher latitudes, underscoring the importance of optimizing light and activity timing for hematological health.”

 

Authors should include critical appraisal tool for their study which will improve the trasparency. Either a JBI tool https://jbi.global/critical-appraisal-tools or a ROBINS-I tool. This enhances the quality of the study. They can include it as a table or supplementary material.

 

A3 Thank you for your suggestion to enhance the transparency and quality of our study by incorporating a critical appraisal tool. We agree that applying such a tool can strengthen the methodological rigour and allow readers to assess potential biases systematically.

 

Given that our study is an analytical cross-sectional design examining associations between circadian parameters and hematological markers, we applied the JBI Critical Appraisal Checklist for Analytical Cross Sectional Studies (https://jbi.global/critical-appraisal-tools). This tool evaluates key aspects such as sampling, exposure measurement, outcome assessment, and confounding control, which align well with our protocol. We now provided new supplementary table (Supplementary Table S2) to the manuscript. We now added to Discussion the following text:

“To further enhance transparency, we have included a completed JBI Critical Appraisal Checklist for Analytical Cross Sectional Studies as Supplementary Table S2, affirming the methodological robustness of our study.”

 

Why have authors dived deeper on the reticulocyte count or number of immature cells if there is a theory that it affects Hb production/erythropoesis. Please expand on this or re-do analysis to include reticulocyte count.

 

A4: Thank you for your comment. Our results linking circadian blue light exposure to hematological markers (HGB, MCH, RDW-CV) were unexpected, as we did not initially hypothesize effects of light on hemoglobin or erythropoiesis. We used routine admission CBC data (standard for freshmen screenings), which does not include reticulocyte count—a specialized test for assessing immature cells and erythropoiesis.

We acknowledge this limitation, especially given theories on circadian HSC rhythms and iron homeostasis influencing erythropoiesis. re-analysis is not feasible due to data constraints, but your suggestion is valuable for future studies. We now added to the Discussion / Study Limitations:

“While our findings link circadian BLE to erythrocytic variables, the absence of reticulocyte counts in routine CBCs precludes direct exploration of erythropoiesis impacts. Future studies should incorporate reticulocyte analysis to investigate direct effects on erythropoiesis, potentially leveraging known circadian human stem cells and iron pathways.”

 

Was electrophoresis done in the study? Will that help? Please include this info in the discussion.

 

A5: We appreciate this comment. Electrophoresis was not performed in this study. As stated in the answer to the previous question, our analysis utilized routine CBC parameters. While electrophoresis offers detailed separation of hemoglobin components, it falls outside the scope of standard CBC and our initial exploratory protocol focused on circadian influences. However, considering our unexpected findings, future research could benefit from incorporating electrophoresis to enhance mechanistic understanding of hemoglobin variations and their circadian associations.

We also added in limitations section right next to above stated para the following text:

“Furthermore, as electrophoresis was not performed due to reliance on routine CBC data, deeper analysis of hemoglobin profiles was not possible, though it could offer broader insights into hematological responses in future research.”

 

Many of the terms used in the article may be new to the readers unfamiliar with your research. Like what is acrophase? New readers may not be aware of these terms. I recommend to include a glossary of such terms as a supplementary file

 

A6: Thank you for this suggestion. As requested by an another reviewer we now provided List of Abbreviations. In addition, we also provided a list of terms as follows:

 

Acrophase: Timing of the peak in a circadian rhythm.

Amplitude: Magnitude of variation in a circadian rhythm, peak-to-trough difference (e.g., daily light fluctuation).

Chronotype: Individual’s natural preference for morning or evening activity

Actigraphy: Non-invasive wrist-worn device monitoring movement, sleep, and light exposure for objective data.

 

Discussion does a good job at what other studies "were about" but lacks in clarity of what other studies "found". Revisions on these aspects should be made.

 

A7: We appreciate this comment from are a reviewer. We now made several additions and clarification in Discussion section as follows:

“Currently, there is a limited scope of studies characterizing the circadian rhythms of CBC components as evident from a recent review (Busza et al., 2024), particularly in relation to environmental light cues, which underscores the novelty of our investigation into how circadian light hygiene modulates these variables.”

“Our previous research at high latitudes found that seasonal changes in circadian light hygiene are associated with changes in morning cortisol with higher values linked to polar seasons and later timing of physical activity and light exposure [28], altered morning lipids (higher low density lipids-cholesterol being linked to elevated light at night, while higher high-density lipids-cholesterol being linked to earlier timing of light exposure) [28–30], and higher clock gene (NR1D1) expression with more abundant light exposure [30]. All these seasonal were coupled with delayed phase melatonin phase and reduction is its relative amplitude [28].”

“Hilderink et al. [32] found small within-subject biological variation for MCH (CVI = 0.8%) and RDW (CVI = 0.37%), indicating stability across time points, and no diurnal fluctuations in healthy populations.”

“...robust light cues, such as a large NA BLE and an earlier BLE acrophase may entrain circadian rhythms of key metabolic regulators like NR1D1 [30,33], as evidenced by studies showing NR1D1 upregulation in response to bright light exposure [30].”

“…it has been previously established that mouse hematopoietic stem cells (HSCs) and their progenitors exhibit robust circadian rhythms, with peak activity occurring approximately 5 hours after light onset [13], leading to higher stem cell mobilization.”

“Future research should prioritize longitudinal studies to establish causality. Multi-month prospective cohorts tracking circadian LE/BLE patterns alongside serial CBC components in populations prone to light disruption (e.g., shift workers, high-latitude residents) can reveal long-term effects on erythropoiesis and anemia risk. Specifically timed bright blue-enriched light to optimize BLE may directly test whether improved circadian light hygiene enhances erythrocytic indices and NR1D1-mediated heme regulation. Mechanistic studies incorporating biomarkers like melatonin and ferritin are also warranted to elucidate photic cue-hematopoiesis pathways.”

 

 

Conclusion is written in a way that keeps everything ambiguous. It should be written in the greatest clarity since authors rely on it. If conclusion is written in a confusing manner, readers will be repulsed and avoid reading the entire article. It should be worded conclusively, "Circadian hygiene improved Hb although <very brief 1-2 limiting points>".

Feel free to use your own language but I believe the conclusion needs to be entirely modified.

 

A8: We appreciate this insightful suggestion. Conclusion was now updated as follows:

“This research reveals significant associations between circadian light exposure patterns and hematological variables, particularly MCH, HGB and RDW-CV, in young adults during light deficient fall season. Larger NA BLE and earlier BLE acrophase were linked to higher MCH and smaller RDW-CV, and NA BLE was independently associated with higher HGB. These findings underscore the supportive role of circadian light hygiene in maintaining hematological health, providing a foundation for future investigations into light’s impact on hematopoiesis.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for the revisions and no further concerns.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed concerns and also improved quality assessment, discussion, limitations and conclusion. I am very pleased with the edits